The present invention is directed to plants and the genetic engineering thereof. More particularly, the present invention is directed to the transformation of plant chloroplasts with foreign DNA.
Most strategies for gene transfer in plants involve the introduction of foreign DNA into protoplasts to enable its integration into the nuclear genome (1-3). However, many of the economically important gene products (e.g., the protein conferring atrazine resistance) either are chloroplast encoded or, if they are nucleus encoded, are functional within the chloroplasts (e.g., enol-pyruvylshikimate-phosphate synthase, which confers resistance to glyphosate) (4) or mitochondria (e.g. aryl acylamidase, which confers resistance to propanil) (5, 6). Furthermore, the 1000-fold higher copy number of chloroplast genes relative to nuclear genes (7-9) makes feasible the introduction of multiple copies of foreign genes into plant cells, should the foreign genes become stably integrated into the chloroplast genome.
To obtain gene transfer into chloroplasts, the isolation of intact organelles capable of efficient uptake, transcription, and translation of foreign DNA is essential. As a first step towards achieving this goal, Daniell and Rebeiz isolated plastids from dark-growth cucumber cotyledons (etioplasts) capable of synthesis of protocholorophyllide (10) and chlorophyll (11-13) at extremely high rates. Also, etioplasts that had been loaded with prothylakoid proteins by treatment of etiolated cucumber cotyledons with hormones (14) converted prothylakoids into macrograna when illuminated in a cofactor-enriched medium (15). Daniell and colleagues also demonstrated the development of electron transport coupled to photophosphorylation in concordance with the synthesis of required polypeptides in isolated etioplasts (16, 17). Finally, they also observed linear biosynthesis of pigment and translation of endogenous messages for 8 hr. (18). These observations collectively establish that etioplasts of cucumber cotyledons are both metabolically very active and unusually stable in their capacity for protein synthesis, marking them as exceptional targets for gene incorporation and expression.
The present invention provides a method for transforming plants with foreign DNA by introducing foreign DNA into chloroplasts. Thus, in one embodiment, the present invention provides a chloroplast from a plant comprising foreign DNA. In another embodiment, the present invention is directed to a method of providing for the uptake of foreign DNA into a chloroplast comprising incubating said chloroplast with a chelating agent that chelates metal ions employed by chloroplast nucleases to provide nuclease-inactivated chloroplasts; and incubating said nuclease-inactivated chloroplasts with foreign DNA whereby said foreign DNA is taken up within the chloroplast membrane.